Combination HAART therapy, which predominantly targets the activities ofthe HIV-1 reverse transcriptase, protease, antj integrase enzymes, works in the majority of cases to inhibit HIV-1 replication and slow or stop the development of AIDS in infected patients. Despite the resounding success of HAART, drug resistance arises in a significant number of treated patients. There is therefore a continual need to develop so-called multi-generational drugs that work in the face of viral drug resistance mutations, as well as new compounds that inhibit HIV-1 through entirely novel means. Recent years have highlighted the interactions between host cellular proteins and viral proteins as novel drug targets, and this grant application previously established the critical role that the interaction between the cellular LEDGF/p75 protein and integrase plays in directing HIV- 1 to integrate into active genes. Ongoing work will ascertain the mechanisms of LEDGF/p75 binding to cellular chromatin and details ofthe interaction between LEDGF/p75 and the viral pre-integration complex. Work conducted during the current funding cycle moreover clarified that small molecule allosteric integrase inhibitors that compete with LEDGF/p75 for binding to integrase rather remarkably inhibited HIV-1 replication at the point of viral core maturation, an unprecedented finding for inhibitors of integrase catalytic function. Such compounds will now be used to further clarify the role of LEDGF/p75 in the virus replication cycle. The work will also determine the roles of novel integrase binding proteins in HIV-1 replication and the division of labor among integrase protomers in the pre-integration complex, which in turn could lead to new avenues of antiviral inhibitor development. The grant recently identified gain-of-function mutants in the HIV-1 integrase that enhanced significantly the transduction of target cells that express reverse charged, complementary LEDGF/p75 proteins, which opened the avenue to targeted gene therapy vectors in human cells. Ongoing work will continue to optimize retroviral DNA integration targeting through the modification of host cell proteins. The successful completion of these studies will refine the mechanisms of action of known targets of antiretroviral drugs, discover new targets for inhibitor development, and invent ways to introduce transgenes. RELEVANCE (See instructions): The work will clarify the mechanism of action of known antiretroviral drugs and uncover new targets for small molecule inhibitor development. Technologies to direct the integration of retroviral vectors will improve the safety of genetic transduction.